Renal type A intercalated cells (A-ICs) actively secrete protons and are key players in the regulation of blood pH. However, the mechanisms by which A-ICs detect sysmetic pH variations are poorly understood. Proton secretion is achieved by the V-ATPase, and severe diseases such as distal renal tubular acidosis and kidney stones result from dysfunctional V-ATPase. While searching for potential acid/base sensors, we found that A-ICs and similar proton-secreting cells in the epididymis (clear cells; CCs) sense luminal bicarbonate via sAC, a bicarbonate-activated adenylyl cyclase. This proposal now examines how A-ICs sense urinary pH in addition to bicarbonate to regulate proton secretion. Some purinergic receptors were proposed as pH sensors, and Aim 1 will test the hypothesis that A-ICs sense urinary pH variations via these receptors. The proposed experiments are based on our new data showing that luminal ATP stimulates proton secretion by CCs. We will identify P1 and P2 receptors that are exclusively expressed in A-ICs, and we will examine their role in regulating V-ATPase-dependent proton secretion. We will also examine ATP secretion by A-ICs and the role of CFTR in this process. A-ICs isolated by FACS from B1-EGFP mice, the C11 intercalated cell line, and microdissected OMCDs will be examined using complementary imaging, biochemical and electrophysiological procedures. Aim 2 will explore the possibility that the V- ATPase itself might be part of the pH-sensing property of A-ICs. These experiments are based on exciting data showing that the V-ATPase a2 subunit is a pH sensor involved in intracellular targeting events. We will examine whether the plasma membrane a4 is a pH sensor that regulates V-ATPase via association of its cytosolic V1 with its transmembrane V0 domains. We will use the epididymis as a model system in which luminal pH can be modulated in vivo. V-ATPase assembly/disassembly will be examined using confocal microscopy, EM, co-IP and cell fractionation. We will translate our findings to A-ICs by using FACS isolated A-ICs and OMCDs perfused in vitro. These experiments will contribute to our better understanding of renal luminal acidification, which is central to the maintenance of blood pH within a very narrow viable range.